The tomato crop in Spain covers 59,300 hectares and production comes to more than 4.3 million metric tonnes per year and Spain is the fourth leading tomato-producing country, only after the United States (California), China and Italy (www.magrama.gob.es/estadistica/pags/anuario/2011/AE_2011_13_06_27_01.pdf). Most of the tomato crop is grown in. Extremadura (73%), Andalusia (13%) and the Ebro Valley (10%) (www.navarraagraria.com/n184/artoma11.pdf). Tomatoes are also a major crop in Portugal, with 15,300 hectares planted and a production of over 1.1 million tonnes (www.ine.pt/ine_novidades/Estatisticas_Agricolas_2011/index.html#). One of the most important pests of tomato is the tomato fruit borer, Helicoverpa armigera (Lepidoptera: Noctuidae) (Torres-Vila et al., 2003). Worldwide, few pests cause as many economic losses as the noctuid H armigera (Cunningham et al., 1999; Reed and Pawar, 1982). In Spain H. armigera has been a key pest on crops such as cotton and corn and, for over a decade, this pest has been gaining importance in the greenhouses in Southeastern Spain, from there spreading to the rest of the regions of Spain and to Portugal (Torres-Vila et al., 2003). It is currently regarded as the most serious phytophagous species in a large portion of field-grown tomato crops in the Mediterranean region (Torres-Vila et al., 2003). The larvae can attack crops in all stages of growth, although the flowering stage is preferred by females for laying their eggs. They prefer the parts of plants with high nitrogen concentrations, such as the reproductive structures (flower and fruit) and the growing tips, so infestation has a direct effect on harvests. Furthermore, the species is highly polyphagous, highly mobile, highly fecund and multivoltine, so population levels can change rapidly both in space and in time. Damage thresholds used by tomato canning company quality controls are between 2 and 5% of harvested tomatoes. If larvae are present, the threshold drops to 0-2% (Torres-Vila et al., 2003). These quality thresholds make plain the need for an efficacious pest control method against H armigera. 
H. armigera control ordinarily takes the form of applying chemical insecticides (Torres-Vila et al., 2003). However, indiscriminate use of synthetic insecticides has given rise to a variety of problems, such as increased production costs, the development of resistance to the various active ingredients, destruction of useful fauna, lower quality owing to higher chemical residues on fruits and fruit products (Torres-Vila et al., 2000). This has spurred the search for other control methods, including virus and other entomopathogenic microorganisms (Moscardi, 1999).
The Family Baculoviridae (baculoviruses) is the most widely studied of all those infecting insects, and is useful to man in that these viruses possess highly desirable traits as bioinsecticides, namely high pathogenicity, compatibility with pests' natural enemies, high specificity (they specifically target arthropods) (Gröner, 1986), long-lasting persistence where protected from ultraviolet light, high horizontal transmission and hence the ability to cause epizootic outbreaks (Caballero et al., 1992; Gelernter and Federici, 1986). In addition, they can be formulated just like synthetic chemical insecticides, are fully compatible with chemical insecticides and can be applied using conventional equipment (Cherry and Williams, 2001). Baculovirus isolates have been collected from around the world and characterized biologically and biochemically (Gelernter and Federici, 1986; Caballero et al., 1992; Hara et al., 1995). Some have already been registered as insecticides in different parts of the world and are being used for pest control (Moscardi, 1999).
Baculoviruses were previously classified into two genera on the bases of viral occlusion body (OB) morphology: Nucleopolyhedrovirus, in which the occlusion bodies are formed from polyhedrin in the shape of irregular polyhedrons and Granulovirus (GV), in which the occlusion bodies are formed from granulin and are granular in shape (Theilmann et al., 2005). However, a more recent, phylogenetically based (genome homology) classification divides the Family Baculoviridae into four genera: Alphabaculovirus (lepidopteran-specific nucleopolyhedroviruses [NPVs]), Betabaculovirus (lepidopteran-specific GVs), Deltabaculovirus (dipteran-specific NPVs) and Gammabaculovirus (hymenopteran-specific NPVs) (Jehle et al., 2006).
Baculoviruses have a double-stranded circular DNA genome enclosed within a protein capsid to form the nucleocapsid, which in its turn is surrounded by a trilaminar envelope composed of a protein layer between two lipid layers acquired during replication of the virus, forming the virion (Caballero et al., 2001). This lipoprotein membrane can be acquired in one of two ways, therefore giving rise to two types of virion. If the nucleocapsids remain in the same cell in which they were formed, they acquire a membrane synthesized de novo, giving rise to occlusion-derived virions (ODV), which are subsequently embedded in a matrix mainly formed by a single protein, giving rise to an occlusion body (OB). However, after synthesis, other nucleocapsids migrate and leave the host cell, acquiring a membrane from the host cell's cytoplasmic membrane as it crosses the cell membrane at specific points where a virus-encoded glycoprotein has been inserted (GP64 or F-protein, depending on the virus). These virions are budded virus (BVs) that move freely in the host's hemocoele and are responsible for spreading the infection to cells in various other tissues. In this stage all baculoviruses synthesize large quantities of polyhedrin (in the case of nucleopolyhedrovirus or NPV) or granulin (in the case of granulovirus or GV), which crystallize to form a matrix or occlusion body (OB) in the form of irregular polyhedrons (polyhedrin) or granules (granulin). For this reason, OBs made of polyhedrin are also known as polyhedra, while those made of granulin are also known as granules. In the final stages of infection, after three to six days, larvae die with large numbers of occlusion bodies that are readily observable under the optical microscope. The infection results in degradation of the larval tegument, releasing millions of occlusion bodies, which contaminate the leaves of the plants and serve as inocula for new infections in other susceptible hosts (Caballero et al., 2001).
Therefore, baculoviruses have two types of morphologically and functionally different virions, or infectious viral particles. ODVs are present in all known baculoviruses and are the infectious particles responsible for primary infection of the epithelial cells of the midgut (alimentary canal) and hence are responsible for horizontal transmission of the virus between susceptible individuals. BVs, in their turn, always contain a single nucleocapsid and are morphologically identical in all cases (FIG. 1A). The BVs are infectious particles responsible for producing the secondary infection, spreading the infection to susceptible organs and tissues of the hemocoele of the host and in in vitro cell cultures (Caballero et al., 2001). The occlusion bodies of NPVs contain several ODVs, whereas granules or GVs contain only one. Morphologically, there are two different types of nucleopolyhedrovirus ODVs, one type gives rise to single nucleopolyhedrovirus (SNPV), having a single nucleocapsid per virion, the other to multiple nucleopolyhedrovirus (MNPV), having from one to several nucleocapsids per virion (FIG. 1B).
Occlusion bodies, both polyhedrons and granules, protect the virions, keeping the virus infectious outside the host. The OBs are capable of surviving in the environment for long periods in places protected from ultraviolet light, are water insoluble, are resistant to putrefaction and to disintegration by chemical agents and are also resistant to such physical treatments as freezing, desiccation and lyophilization. In contrast, occlusion bodies are soluble in alkaline solutions like those found in the digestive tract of certain insects (pH 9-11), thereby releasing the ODVs to initiate an infection (Caballero et al., 2001).
Baculoviruses have been isolated from more than 500 insect species, mainly in the Order Lepidoptera, including many of the most important agricultural pests. Besides considerable interspecific diversity, baculoviruses also exhibit high intraspecific diversity, as has been demonstrated by characterization of different geographical isolates of the same virus and of single isolates, with wild isolates often comprising different genotypic variants. Viral DNA analysis with restriction enzymes is commonly used to differentiate and characterize both isolates and the genotypes present in a single isolate, as this procedure results in characteristic profiles for each isolate or genotype (Erlandson et al., 2007; Figueiredo et al., 1999; Harrison and Bonning, 1999).
Genome variation between different isolates and genotypes of the same virus can give rise to significant differences in their insecticidal characteristics, such as pathogenicity, defined as the amount of inoculum needed to kill a percentage of the population, virulence or the speed with which it kills the insects and viral productivity. Host range, occlusion body size and larval liquefaction are other phenotypic traits that may be affected (Cory et al., 2005; Harrison et al., 2012). Knowing the intrapopulation diversity of baculoviruses therefore has special importance when it comes to designing bioinsecticides, the active ingredients of which should include the strains or genotypes that have the greatest insecticidal potential. Furthermore, local insect populations are known to be more susceptible to native isolates of the virus (Barrera et al., 2011; Bernal et al., 2013a), making it appropriate to select a virus isolate having the same geographical origin as the populations to be controlled.
H. armigera larvae are naturally infected by a nucleopolyhedrovirus known by the abbreviated name of HearSNPV (Helicoverpa armigera single nucleopolyhedrovirus, genus Alphabaculovirus). This is a single nucleopolyhedrovirus (SNPV) that also infects the larvae of other members of the genera Helicoverpa spp. and Heliothis spp., for instance, Helicoverpa zea larvae. Characterization has been performed on HearSNPV isolates from different regions around the world, such as China and Kenya (Chen et al., 2001; Ogembo et al., 2005). Isolates of this virus have also been obtained from Spain and Portugal (Figueiredo et al., 1999, 2009), where it causes natural epizootic outbreaks in H. armigera populations. Several isolates of this virus have been characterized to date, with most studies being carried out on:                Two pure genotypes from China, the genomes of which have been completely sequenced, HearSNPV-G4 (Chen et al., 2001) and HearSNPV-C1 (Zhang et al., 2005), which will be referred to in the rest of this specification using the abbreviations HearG4 and HearC1, respectively. Guo et al. (2006) compared the biological activity of these two genotypes. On the basis of the concentration-mortality relationship, HearC1 turned out to be 2.8 times more pathogenic than HearG4 against third-instar larvae of an H. armigera population from China. In addition, larvae infected with HearC1 died nine hours sooner than larvae infected with HearG4. Zhang et al.'s 2005 article compared the genomes of these two genotypes and found the nucleotide sequences to be 98.1% identical. Comparing the two genomes revealed four variable regions between the two genotypes, homologous regions 1, 4 and 5 (hr1, hr4 and hr5) and the bro-b region. Homologous regions (hrs) are intergenic regions present in many baculoviruses and located many times along the genome. They are characterized by the presence of multiple imperfect repeat sequences. The genome of HearSNPV contains five homologous regions. FIG. 1 in the article by Chen et al. (2000) shows the restriction profiles for the BamHI, Bg/II, EcoRI, HindIII, KpnI, PstI, SacI and XhoI restriction endonucleases (FIG. 2 in this application). Table 1 in that article sets out the estimated sizes of the restriction fragments generated by each of the said restriction endonucleases (REN) (Table 1). The complete genomes of HearG4 and HearC1 are available in the GenBank database under accession numbers AF271059 and AF303045, respectively. The HearG4 genotype is currently commercially available for controlling H. armigera on cotton crops in China (Zhang, 1994).        
TABLE 1Estimated sizes of HearG4 fragments generated by digestion with BamHI, BglII, EcoRI, HindIII, KpnI, PstI, SacI and XhoI and estimated total genome size (Chen et al., 2000).FragmentBamHIBglIIEcoRIHindIIIKpnIPstISacIXhoIA37.324.514.122.255.539.065.036.5B31.818.513.916.534.236.822.334.6C14.415.89.814.723.632.319.320.0D14.014.89.112.89.811.89.711.0E12.713.79.011.66.16.19.410.9F7.712.16.810.80.93.44.47.0G3.97.16.410.20.64.4H3.35.96.010.13.5I1.94.96.07.32.2J1.84.35.86.5K1.33.45.63.2L2.64.72.7M2.54.61.5N4.5O4.4P4.3Q3.7R3.3S3.1T1.7U1.0V0.8W0.5X0.5Y0.5Total130.1130.1130.1130.1130.1130.1130.1130.1                An isolate from Kenya, HearSNPV-NNg1, referred to here as HearNNg1, the genome of which has also been sequenced completely (Ogembo et al., 2009). HearNNg1 was selected by Ogembo et al. (2007) as the isolate having the best attributes for development as a bioinsecticide against H. armigera larvae in Japan. Against third-instar larvae HearNNg1 was between 3.2 and 82.6 times more pathogenic than the other isolates studied and 311.5 times more pathogenic than the Chinese isolate HearG4. In addition, NNg1 killed third-instar H. armigera larvae between 0.4 and 1.8 days sooner than the other isolates and 4.3 days sooner than the HearG4 genotype. FIG. 1 in that article sets out the restriction profiles for the isolates characterized using Bg/II and XbaI endonucleases (FIG. 3 in this application). Table 2 in that same article sets out the estimated sizes of the restriction fragments generated for the different isolates digested by Bg/II, XbaI and HindII endonucleases (Table 2).        
TABLE 2Estimated sizes of fragments of HearNNg1 (NNg1) and other isolates from SouthAfrica (NS2), Kenya (NMa1), Zimbabwe (NZ3), Thailand (NT1) and China (G4) generated bydigestion with BglII, XbaI and HindIII and total estimated genome size (Ogembo et al., 2007).BglIIXbaIHindIIIFragmentNNg1NS2NMa1NZ3NT1G4NNg1NS2NMa1NZ3NT1G4NNg1NS2NMa1NZ3NT1G4A23.725.525.523.723.725.514.214.214.214.214.214.222.622.622.622.622.622.6B18.718.718.718.718.718.713.013.013.013.013.013.014.517.117.117.114.514.5C15.315.315.315.315.315.311.911.911.911.912.411.91313.513.513.514.514.5D15.015.015.015.013.315.010.610.610.610.611.910.6111313131313E13.313.313.313.312.413.39.39.39.39.310.69.310.81111111111F12.412.412.411.510.712.49.19.17.29.19.39.110.710.810.410.810.810.7G10.710.710.710.79.46.97.27.26.27.29.17.210.410.41010.410.410.4H9.46.96.96.98.85.86.26.26.16.26.26.210108.2101010I4.34.34.34.36.95.06.16.15.96.15.95.97.78.27.57.57.57.5J3.33.33.33.34.34.35.95.95.75.85.75.87.57.543.36.76.7K2.72.62.63.23.33.35.75.75.55.75.55.76.73.33.32.643.3L2.51.31.32.62.72.65.55.55.45.54.05.53.32.631.92.62.6M———1.32.52.55.45.44.85.43.64.02.61.52.61.51.51.5N3.44.84.64.83.33.31.51.5———O3.24.63.63.43.23.2P3.14.43.23.22.12.5Q1.93.61.61.61.62.1R1.63.11.21.21.21.9S1.21.91.11.11.11.6T1.11.61.01.01.01.3U1.01.2———1.2V1.1———1.1W1.0————Total129.3131.3129.3129.8132130.6137.4126.6122.1126.2124.9126.6131.5132.3127.7125.2132.2131.4
Furthermore, the article by Ogembo et al. (2009) compares the HearNNg1 genome with the genomes of the Chinese genotypes HearC1 and HearG4, and with the genome of Helicoverpa zea single nucleopolyhedrovirus (HzSNPV). The greatest differences of the NNg1 genotype with respect to the HearC1, HearG4 and HzSNPV genomes were in the homologous regions (hrs) and in the bro genes, as occurred in the comparison of the HearC1 and HearG4 genomes. The whole HearNNg1 genome is available in the GenBank database under accession number AP010907.                An Australian isolate, HearSNPV-Aus, which will be referred to in this specification by the abbreviation HearAus, the genome of which has been completely sequenced and is available in the GenBank database under accession number JN584482.        Seven isolates from the Iberian Peninsula: five from Spain, HearSP1, HearSP2, HearSP4, HearSP7 and HearSP8, and two from Portugal: HearPT1 and HearPT2 (Arrizubieta et al., 2014; Figueiredo et al., 1999, 2009). Figueiredo et al. (1999) selected the HearSP1 isolate as the one having the best insecticidal properties, in that it was two times more pathogenic than HearSP2 against second-instar larvae from a Portuguese population. Subsequently, a new study by Figueiredo et al. (2009) found that the HearSP7, HearPT1 and HearPT2 isolates exhibited the best bioinsecticidal attributes, though the study did not include the HearSP1 isolate. A recent study performed at our laboratory comparing all these Iberian Peninsula isolates selected HearSP1 as having the best insecticidal attributes against H. armigera, as it had the same pathogenicity as the other isolates considered, but it was more virulent and was also one of the most productive in terms of the number of occlusion bodies produced in each infected insect (Arrizubieta et al., 2014). FIG. 1B in the article by Figueiredo et al. (2009) presents the Bg/II restriction profiles for the Spanish isolates HearSP1, HearSP2, HearSP3, HearSP4, HearSP7 and HearSP8 and Portuguese isolates HearPT1 and HearPT2 (FIG. 4A in this application). FIG. 1 in the article by Arrizubieta et al. (2014) presents the EcoRI profiles for the HearSP1, HearSP2, HearSP4, HearSP7, HearSP8, HearPT1, HearPT2 and HearG4 isolates (FIG. 4B in this application) and Table 1 in that article listed the restriction fragment sizes (Table 3).        
TABLE 3Estimated fragment sizes for HearSP1, HearSP2, HearSP4, HearSP7, HearSP8, HearPT1, HearPT2 and HearG4 and actual fragment sizes for HearG4 generated in silico (G4*) froma sequence (AF271059) generated by digestion using EcoRI and total estimated genome sizes(Arrizubieta et al., 2014).HearSNPV isolateFragmentSP1SP2SP4SP7SP8PT1PT2G4G4*A13.413.413.413.213.413.413.414.314.13B10.713.210.710.010.710.710.713.413.45C9.310.79.39.39.09.39.310.110.15D9.29.39.29.08.29.09.29.09.05E8.29.28.28.27.58.28.26.66.64F7.17.17.17.16.37.57.56.46.36G6.36.36.36.36.06.36.36.36.29H6.06.06.06.05.96.06.06.05.99I5.95.95.95.95.85.95.95.85.84J5.85.85.85.85.85.85.85.85.84K5.85.75.85.85.75.85.85.75.67L5.75.35.75.74.95.35.74.84.75M5.34.94.94.94.64.95.34.64.58N4.94.64.64.64.44.64.94.44.42O4.64.44.44.44.44.44.64.44.40P4.44.44.44.43.34.44.44.14.14Q4.43.33.33.33.03.34.43.73.68R3.33.03.03.02.83.03.33.43.36S3.02.82.82.81.72.83.03.03.0T2.81.71.71.71.01.72.82.82.83U1.71.01.01.01.01.01.71.71.74V1.01.01.01.00.81.01.01.51.48X1.00.80.80.80.81.01.01.00Y0.80.80.80.78Z0.50.48a0.4———————0.45b0.4———————0.41c0.3———————0.31d0.18———————0.18e0.02———————0.02Total132.4129.8125.3124.2116.2125.1131.0129.6131.4
The difference in the number of different genotype fragments with the number generated in silico for the HearG4 genotype is attributable to the fact that its genome has been completely sequenced, making it possible to detect small fragments not visible on the REN profiles and hence impossible to detect by banding pattern analysis. In the case of HearSP1, small fragments were detected by PCR amplification and sequencing the amplified fragments using designed primers on the ends of the cloned fragments (Arrizubieta et al., 2014).
After selecting appropriate active material and before a bioinsecticide is marketed, field trials have to be performed to verify that it is efficacious in the conditions in which it will be applied, given that its efficacy in the field may vary from that recorded under controlled conditions in the laboratory. However, to be able to treat large areas of crop in order to carry out the field trials, large amounts of occlusion bodies are required, making it necessary to develop a system for mass producing the virus. The method currently employed for mass production of most baculoviruses is in vivo production in permissive hosts (Kalia et al., 2001; Lasa et al., 2007). This method consists of feeding susceptible larvae an artificial diet contaminated with a suspension of occlusion bodies on the surface. Certain essential aspects of this method, such as the artificial diet for the insect or mass breeding methods have to be developed specifically for each host-pathogen system (Lasa et al., 2007). Furthermore, a HearSNPV production system involving both in vivo and in vitro production has been developed in the United States (U.S. Pat. No. 7,521,219 B2). This method consists of first multiplying the virus in H. armigera larvae and then performing a limited number of serial passages in cells to obtain large amounts of occlusion bodies.
Since H. armigera larvae are developing resistance to synthetic chemical insecticides with ever greater frequency, the amount that has to be applied for these insecticides to achieve the sought-after effect is gradually increasing. Owing to the large land area given over to growing tomatoes in the Iberian Peninsula, this is turning into a problem with huge negative impacts for growers, consumers and the environment.
Contamination of soils, aquifers and other natural areas; their effects on other living organisms; and higher production costs of agricultural products coupled with lower product quality represent serious threats to various strategic sectors in the Iberian Peninsula. In view of the resistance to synthetic chemical insecticides developed by H. armigera larvae, there is interest in fostering the availability of an alternative that combines good insecticidal attributes with a very narrow host range to avoid targeting natural enemies and other beneficial organisms, for example, a biological control agent. One especially desirable agent of this kind would be an efficient control method sufficiently potent to counter the threats and predicaments posed by H. armigera in the Iberian Peninsula. In addition to being highly efficacious against pests in the Iberian Peninsula, there is also a need for an efficient production method, so that production costs and the amounts of insecticide to be applied do not make it uncompetitive by raising costs.
This invention provides an effective solution to these problems.